This proposal describes a research plan to biosynthesize HSAF (dihydromaltophilin), a broad spectrum antifungal natural product with a new mode of action. HSAF was isolated from Lysobacter enzymogenes C3, a bacterium used in the biological control of fungal diseases in agriculture. HSAF exhibits potent activities against a wide range of fungal species, including the life-threatening human pathogen, Aspergillus fumigatus, and shows a novel mode of action by disrupting the polarized growth of filamentous fungi. The antifungal activity of HSAF is mediated through a distinct ceramide synthase that is only found in the filamentous fungi. The disruption of this ceramide synthase gene leads to the depletion of a group of sphingolipids that are probably required for polarized growth of fungi. Fungal sphingolipids are structurally distinct from mammalian sphingolipids and represent a new target for antifungal drugs. HSAF has a complex chemical structure, including a tricyclic system fused to a macrolactam containing a tetramic acid, which is different from any existing antifungal drugs. Thus, HSAF has an unprecedented mode of action and a new chemistry, which are criteria for new antifungal agents. However, chemical total synthesis of HSAF is not feasible for commercial production due to its highly complex structure. Biosynthesis via microbial fermentation is a viable approach, which requires an understanding of the biosynthetic mechanism. The long-term goal is to exploit the potential of the tetramic acid-containing macrolactams as a new class of antifungal drugs. The objective of this project is to determine the mechanism for the formation of HSAF tetramic acid and macrolactam and to test the feasibility of HSAF biosynthetic engineering in L. enzymogenes C3. Chemical total synthesis of HSAF is not feasible for commercial production due to its highly complex structure. Biosynthesis via microbial fermentation is a viable approach, which requires an understanding of the biosynthetic mechanism. The formation of tetramic acid and the macrolactam is the key step in HSAF biosynthesis, and the feasibility test for biosynthetic engineering in the host is also essential for the long-term goal. Two specific aims are outlined in the proposal to realize the objective of the project. The Specific Aim 1 is to determine the reactions catalyzed by the nonribosomal peptide synthetase (NRPS) of HSAF synthase. A key feature of HSAF is that it has two amides that are formed from the same amino acid (ornithine). This is distinct from other tetramic acid-containing polyketides. The Specific Aim 2 is to replace the NRPS module to test the feasibility of synthesizing new HSAF analogs in L. enzymogenes. HSAF belongs to a group of natural products with unique structural features and diverse biological activities, such as the anticancer agents discodermide and cylindramide A from marine sponges and the antiprotozoal agent ikarugamycinfrom Streptomyces. None of these products have been studied for their biosynthetic mechanism. The proposed studies will not only determine the mechanism for a key step in HSAF biosynthesis and test the feasibility for producing new HSAF analogs, but also provide information for the biosynthesis of the other complex natural products. PUBLIC HEALTH RELEVANCE The over reliance on a small set of targets in the current therapeutics for fungal infections in humans has led to the alarming increase of drug resistance, especially in AIDS patients and people using invasive medical devices and implants. HSAF provides a new opportunity to combat the drug resistance because it is an antifungal natural product with a new mode of action and a new chemistry. The proposed studies will help develop strategies for the preparation of this product and its analogs to treat fungal infections.